INTRODUCTION

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-Lactamases (EC 3.5.2.6) are the bacterial enzymes that hydrolyze the -lactam amide bond of -lactam antibiotics, and their production is the most common mechanism of bacterial resistance to these antibiotics. -Lactamases are divided into four classes, classes A to D, on the basis of their primary structures (1). The enzymes can also be classified into two groups on the basis of the differences in their catalytic mechanisms, i.e., serine -lactamases (classes A, C, and D) and metallo--lactamases (class B). Metallo--lactamases have broad substrate specificity, including carbapenems, which are otherwise quite stable to serine -lactamases. A limited number of bacterial species were known to produce metallo--lactamases; therefore, the enzymes were thought to be of little consequence clinically. However, we isolated an R-plasmid-mediated metallo--lactamase gene from Klebsiella pneumoniae DK4 in 1991 (GenBank accession number D29636). This was the first finding of an R-plasmid-mediated metallo--lactamase gene in a member of the family Enterobacteriaceae. On the basis of the nucleotide sequence of the DK4 -lactamase gene, the DK4 enzyme was confirmed to be identical to a metallo--lactamase mediated by the bla_IMP gene located in the chromosome of a Serratia marcescens strain isolated in 1991 (18).

Recently, the bla_IMP gene has been found to be widely distributed in clinical isolates in Japan, including among members of the family Enterobacteriaceae and Pseudomonas aeruginosa isolates (2, 10, 24). Although kinetic parameters for common -lactams were reported (13, 16, 18), little is known about other molecular characteristics of the metallo--lactamases. The study described here was carried out to understand the functional properties of the DK4 (IMP-1) -lactamases by using site-directed mutagenesis.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. K. pneumoniae DK4 is a clinical strain isolated in 1991 in Tokyo, Japan, and its -lactam resistance is mediated by an R plasmid termed RDK4. Escherichia coli 1037 Rif^r (11) was used as the recipient for R-plasmid transfer. E. coli AS226-51 (27) is an ampD mutant of strain C600 and a mutant with an ampC deletion and was used as the host for enzyme preparation. E. coli TG1 (5) was used as the host for the cloned -lactamase gene. E. coli MV1184 (28) was used as a recipient for transformation in the site-directed mutagenesis study. Plasmid pHSG398 (25) was used as a cloning vector that carries a marker for resistance to chloramphenicol. Plasmid pKF18K (Takara Shuzo Co., Ltd.), which has a marker for resistance to kanamycin, was used as the vector for site-directed mutagenesis.

Media, chemicals, and enzymes. Nutrient broth and Drigalski agar (Eiken Chemical Co., Tokyo, Japan) were used as the culture medium and the selective agar for R-plasmid transfer, respectively. For transformation, 2× yeast extract-tryptone (2×YT) broth and yeast extract-tryptone (YT) agar were used. For -lactamase preparation, the bacteria were grown in heart infusion broth (Eiken Chemical Co.) or Terrific broth. Heart infusion agar was used to measure the susceptibilities of the bacteria to -lactams.

Antibiotic susceptibility testing. Bacterial susceptibility to antibiotics was measured by the serial agar dilution method by a previously described procedure (23) and was expressed as the MIC of each drug.

Cloning of -lactamase gene. RDK4 DNA was prepared from E. coli AS226-51/RDK4 by the alkaline lysis method and was digested with a restriction endonuclease, PstI. The digested DNA fragments were ligated into the PstI site of a vector plasmid, pHSG398. The resultant recombinant plasmid DNA was confirmed to have incorporated a 9.7-kb DNA fragment and was designated pDK4-1. The pDK4-1 DNA was retransferred into E. coli AS226-51, which was confirmed to produce a -lactamase with a pI of 8.3, identical to that of the DK4 -lactamase. A 1.2-kb DNA fragment was finally prepared by digestion of pDK4-1 with StyI, and the fragment was confirmed to include the intact -lactamase gene by blunt-ending analysis. The fragment was inserted antiparallel with respect to the lacZ direction into the HincII site of pHSG398. The cloned plasmid DNA was termed pDK4-5. The 1.2-kb DNA fragment was sequenced by the dideoxynucleotide chain-termination method (21) with a BcaBEST dideoxy sequencing kit (Takara Shuzo Co., Kyoto, Japan).

Site-directed mutagenesis. Site-directed mutagenesis was carried out by use of the oligonucleotide-directed dual amber method (9) with a template plasmid clone, pKFDK4. The template plasmid was prepared by insertion of the 1.2-kb DNA fragment containing the -lactamase gene into pKF18K antiparallel with respect to the lacZ direction. The entire mutant gene was sequenced by the dideoxy chain-termination method to confirm the desired exchange in the nucleotide sequence.

Isoelectric focusing. Isoelectric focusing was carried out by use of a model 111 IEF cell (Bio-Rad Laboratories, Hercules, Calif.) and a gel plate containing 5% Ampholine (pH 3.5 to 9.5). The -lactamase activity on the plate was detected by spraying with a chromogenic cephalosporin, FR18419. The -lactamases from the following strains were used as isoelectric markers: Citrobacter freundii GN346, pI 8.9 (22); Proteus mirabilis N29/pCS229, pI 6.9 (20); and E. coli ML1410/RGN823, pI 5.4 (22).

-Lactamase purification and -lactamase assay. E. coli AS226-51 cells carrying the wild-type or a mutant -lactamase gene were grown overnight at 37°C in heart infusion broth or Terrific broth containing a sublethal concentration of kanamycin (50 µg/ml). The preculture was diluted with a 40-fold volume of fresh medium, followed by growth under aeration at 37 or 24°C. In the cases of the bacterial cells carrying the mutant gene with the H28A, H86A, H88A, H149A, and C168A mutations, bacterial growth was carried out at 24°C to achieve a higher yield of the active enzyme than that achieved at 37°C. At the mid-logarithmic phase the bacterial cells were disrupted with a French press in 50 mM MOPS [3-(N-morpholino)propanesulfonic acid] buffer (pH 7.0) containing 0.1 mM zinc sulfate. The disrupted cells were centrifuged for 1 h at 40,000 × g and 4°C, and the supernatant was used for further purification of the enzyme by ion-exchange chromatography on a CM-Sephadex C-50 column equilibrated in MOPS buffer with 0.1 mM zinc sulfate. The enzyme was eluted from the column by a linear NaCl gradient (0.1 to 0.4 M), followed by gel filtration on a Sephadex G-75 column equilibrated with MOPS buffer. The -lactamase in the fractions was detected by cephalothin hydrolysis in 50 mM MOPS buffer (pH 7.0) with 1 mM zinc sulfate or by an immunoblotting technique with the polyclonal antiserum against the DK4 -lactamase. The purity of the enzyme preparation was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme protein was determined from UV absorption at 280 nm with  equal to 44,600 M¹ cm¹. Confirmation was obtained by the Bio-Rad version of Bradford's dye binding assay (4) with bovine serum albumin as a standard.

Protein analysis. The N-terminal sequence was determined by the use of a protein sequencer (model 477A; Applied Biosystems, Foster City, Calif.).

Atomic absorption spectroscopy. The zinc content in the enzyme molecule was determined as follows. All the glassware was washed with 6 N HNO₃ in order to eliminate contamination. Enzyme samples were subjected to 3 days of dialysis against zinc-free 50 mM MOPS buffer (pH 7.0) containing Chelex 100 resin at 4°C with multiple changes of the dialysis buffer, and then the enzyme samples were diluted with the same buffer to provide samples with the expected range of 0.05 to 1.00 ppm of zinc. The preparation was analyzed with a Z-8000 Polarized Zeeman Atomic Absorption Spectrophotometer (Hitachi Ltd.).

Nucleotide sequence accession number. The nucleotide sequence of the DK4 -lactamase gene was entered into the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases under the accession number D29636.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

The DK4 -lactamase gene and its product. K. pneumoniae DK4 exhibited a high level of resistance to extended-spectrum cephalosporins and a moderate level of resistance to carbapenens, i.e., imipenen. The resistance was conjugally transferred to E. coli 1037 Rif^r. The -lactam resistance was due to a -lactamase with a pI of 8.6 and was mediated by an R plasmid termed RDK4. The activity of the DK4 -lactamase was completely inhibited by 10 mM EDTA. The DK4 -lactamase gene in a 9.7-kb DNA fragment was cloned into a plasmid vector, pHSG398, and the recombinant plasmid was termed pDK4-1. From the 9.7-kb DNA fragment, four shorter DNA fragments (of 3.9, 2.6, 2.0, and 1.2 kb) containing the -lactamase gene were prepared, and each fragment was cloned into the plasmid vector (Fig. 1). The recombinant plasmids obtained were termed pDK4-2, pDK4-3, pDK4-4, and pDK4-5, respectively. The complete nucleotide sequence of the 1.2-kb fragment inserted into pDK4-5 was determined. The sequenced region contained an open reading frame, and an amino acid sequence comprising 246 amino acids was deduced. Through determination of the N-terminal amino acid sequence of the purified enzyme protein, a signal peptide with 18 amino acids and a mature enzyme with 228 amino acids were detected.

View larger version (11K): [in this window] [in a new window]   FIG. 1.   Restriction maps of inserts of the clone and subclones carrying the target -lactamase gene.

Dose effect of DK4 -lactamase on resistance to -lactam antibiotics in E. coli cells. During the preparation of the subcloned -lactamase gene, we observed that the -lactamase activity in the E. coli AS226-51 cells was inversely proportional to the size of the DNA fragment inserted into the vector plasmid (Table 1). This phenomenon may be due to the difference in copy number of the plasmid in the host cells because all the -lactamase genes were expressed by their own promoters, and the pDK4 series can be used to provide an understanding of the dose effect of metallo--lactamase production with respect to the level of resistance of the bacterial cells to -lactams. The -lactamase activity of K. pneumoniae DK4 was 0.12 U per mg of bacterial protein, as determined with cephalothin as a substrate. When RDK4 was transferred to E. coli AS226-51 cells, the enzyme activity was 0.098 U per mg of bacterial protein, about 80% of that for the original strain. For the series of E. coli subclones, the MICs for the cells increased up to 8 times and the level of enzyme production increased up to 13 times. The results in Table 1 show quantitatively that the metallo--lactamase contributed to the resistance of the bacteria to all -lactams except the monobactam aztreonam.

View this table: [in this window] [in a new window]   TABLE 1.   Dose effect of DK4 -lactamase on resistance of bacteria to typical -lactams

Characteristics of DK4 -lactamase and its kinetic parameters. The DK4 -lactamase was extracted from the E. coli cells carrying pDK4-5 and was completely purified. From 18 liters of the bacterial culture, 18 mg of the purified enzyme protein was obtained, and its purity was confirmed by SDS-PAGE. The presence or absence of 0.1 mM zinc sulfate throughout the purification processes did not affect the recovery of active enzyme, indicating a tight binding of zinc to the enzyme. The purified enzyme showed a high degree of thermostability, as the enzyme retained its full activity even after 30 min of incubation at 60°C in 50 mM MOPS buffer (pH 7.0) containing 0.1 mM zinc sulfate. The activity was assayed within 30 s of the enzyme reaction at 30°C. The effect of pH on the activity of the enzyme against cephalothin at between pH 5.0 and 8.0 was measured in the following buffers: 50 mM MES buffer with 0.5 M NaCl and 1 mM zinc sulfate (pH 5.0 to 6.5) and 50 mM MOPS buffer with 0.5 M NaCl (pH 7.0 to 80). The -lactamase activity increased concomitantly with pH, such that at pHs 7.0, 6.0, and 5.0 the activities were 75, 49, and 32%, respectively, of that at pH 8.0. The enzyme was found to be unstable at a pH lower than 6.0, and this phenomenon may be attributable to the presence of 2-(N-morpholino)ethanesulfonic acid (MES) (8). Stability at lower pH could be protected by the presence of 1 mM zinc sulfate. We failed to measure activity at a pH higher than 8.0 because of alkaline hydrolysis of the substrate in the reaction mixture.

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View this table: [in this window] [in a new window]   TABLE 2.   Kinetic parameters of DK4 -lactamase for -lactams

The apo-DK4 -lactamase and its restoration. A DK4 -lactamase that was missing the two zincs was prepared by 3 days of dialysis of the EDTA-treated enzyme against zinc-free MOPS buffer. The enzyme, termed apo-DK4, was completely lacking enzyme activity, and its activity was estimated to be less than 0.004% of that of the holoenzyme. The activity of the apo-DK4 enzyme was restored to about 30% of its original activity by the addition of 1 mM zinc sulfate to the enzyme solution. This restoration was hindered in the presence of a sulfhydryl reagent, methylmethane thiosulfonate, suggesting the contribution of a cysteine to zinc binding. The reactivated enzyme had about the same K_m value for cephalothin as that of the native enzyme, and it was therefore thought that about 70% of the enzyme molecules were irreversibly denatured during production of the apoenzyme state.

Functions of His28, His86, His88, His149, His210, and Cys168 as zinc ligands. The alignment of the amino acid sequences of the metallo--lactamases indicated that histidines at positions 86, 88, 149, and 210 were conserved in all the enzymes, and a histidine at position 28 was found in some metallo--lactamases. All of these histidine residues except His28 were estimated to be located at positions close to the zincs by reference to a three-dimensional structure of a metallo--lactamase from B. fragilis (6). Cys168 was also presumed to be situated in the vicinity of the zinc. To establish the functions of these residues in the DK4 -lactamase, these five histidines and the cysteine were individually replaced by an alanine by site-directed mutagenesis, and the mutant genes on the vector plasmid were transformed into E. coli AS226-51.

View this table: [in this window] [in a new window]   TABLE 3.   Zinc contents of DK4 metallo--lactamase and its mutantsa

View this table: [in this window] [in a new window]   TABLE 4.   Kinetic parameters of mutant metallo--lactamases for cephalothin in the presence or absence of 1 mM zinc sulfatea

Function of Asp90 as a general base in the enzyme reaction. Aspartic acid at position 90 is one of the conserved residues in known metallo--lactamases. Concha et al. (6) claimed that Asp90 of a metallo--lactamase from B. fragilis is one of the zinc ligands. The B. fragilis -lactamase with the D90V mutation, in fact, had a lower zinc content than the wild-type enzyme (7). We observed that the k_cat value of the DK4 -lactamase increased with an increase in the pH from 5.0 to 8.0, suggesting the existence of a general base in the reaction. A candidate for the general base was Asp90, which was assumed to be localized to the active-site area. In order to confirm this assumption, Asp90 was replaced by asparagine.

View this table: [in this window] [in a new window]   TABLE 5.   Kinetic parameters of the -lactamase with the D90N mutation for cephalothin and its zinc content

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Effect of pH on the relative kcat values of the wild-type and the D90N -lactamases. The following buffers were used for the assay; 50 mM MES buffers containing 0.5 M NaCl and 1 mM zinc sulfate (pH 5.0 to 6.5) and 50 mM MOPS buffer containing 0.5 M NaCl (pH 7.0 to 8.0). The activity was measured with cephalothin as the substrate. , wild-type enzyme; , enzyme with D90N mutation.